Cells respond to mechanical force in many ways. Recent work has shown that mechanical force applied to cell adhesion molecules can affect the activities of Rho family GTPases, thereby influencing the organization of the cytoskeleton, cell adhesion, migration and many other cellular activities. This project is aimed at elucidating the signaling pathways by which mechanical force affects Rho GTPases, particularly RhoA, under a number of situations. In the first part of this project, we will focus on migrating cells and use shRNA knockdown strategies to identify the GEFs responsible for RhoA activation at the leading edge. Using biosensors for RhoA and relevant GEFs developed by Hahin and Sondeic, combined with the multiplexing imaging strategies of Danuser and i-laiin, we will determine whether GEF activation occurs at the leading edge as a result of integrin engagement or by mechanical force. Our preliminary studies show that mechanical tension on cadherins also activates RhoA and in the second part of the project, working with Hall, we aim to identify the GEFs and signaling pathways involved. Using magnetic tweezers and beads coated with the extracellular domain of E-cadherin we will explore how force leads to the strengthening of cadherin-mediated adhesions. The final section of the project examines how mechanical tension applied to the cell surface affects RhoA signaling in the nucleus. Based on our preliminary work showing that stretching isolated nuclei activates RhoA, we will target the RhoA biosensor to the nucleus so that we can examine RhoA activity in this compartment as mechanical force is applied to the cell surface. We will screen for the GEFs responsible and then examine the consequences of RhoA activation within the nucleus, examining nuclear stiffening and effects on gene expression.